The plasma membrane (sarcolemma) of cardiac myocytes has high levels of Na+-Ca2+ exchange activity. Na+- Ca2+ exchange is an important regulator of intracellular Ca2+ and thus a major determinant of myocardial contractility. Na+-Ca2+ exchange is upregulated and may take on added significance during hypertrophy and heart failure. It is important to investigate the structure and molecular properties of the cardiac Na+-Ca2+ exchange molecule to gain understanding of the role of the exchanger in physiology and pathophysiology. Towards this goal, the specific aims of the proposal are as follows: 1. Structure and Regulation. The Na+-Ca2+ exchanger is regulated by nontransported intracellular Ca2+. The structure of the Ca2+ regulatory site has recently been determined. The goals are twofold: a. Determine the structures of other segments of the intracellular loop of the Na+-Ca2+ exchanger. b. Apply structural information to understand Ca2+ regulation at the molecular level. 2. Helix Packing, Dimerization, and Transport Pathway. The aim is to further investigate the packing arrangement of transmembrane segments within the Na+-Ca2+ exchanger and between exchanger dimers. Mutational approaches will be used to understand the ion translocation mechanism and pathway. 3. Application of Fluorescent Resonance Energy Transfer (FRET) to Study the Na+-Ca2+ Exchanger. The aim is to apply FRET to investigate conformational changes of the exchanger in vitro, in cells, and in a living organism. FRET will also be applied to investigate dimerization of the exchanger. The underlying hypothesis is that increased knowledge of the structure, function, and regulation of the Na+- Ca2+ exchanger molecule will directly lead to a better understanding of the role of the exchanger in physiological and pathophysiological settings. The increased knowledge will perhaps have pharmacological and clinical applications.